1. Field of the Invention
This invention relates generally to the field of protein kinases, oncogenes and oncoproteins and, specifically, to a protein kinase which phosphorylates and potentiates the activity of c-Fos.
2. Description of Related Art
A number of viral and cellular genes have been identified as potential cancer genes, collectively referred to as oncogenes. The cellular homologs of viral oncogenes, the proto-oncogenes or c-oncogenes, act in the control of cell growth and differentiation or mediate intracellular signaling systems. The products of oncogenes are classified according to their cellular location, for example, secreted, surface, cytoplasmic, and nuclear oncoproteins.
Proto-oncogenes which express proteins which are targeted to the cell nucleus make up a small fraction of oncogenes. These nuclear proto-oncoproteins typically act directly as transactivators and regulators of RNA and DNA synthesis. Nuclear oncogene products have the ability to induce alterations in gene regulation leading to abnormal cell growth and ultimately neoplasia. Examples of nuclear oncogenes include the myc, ski, myb, fos and jun genes.
The c-Fos protein, encoded by the c-fos proto-oncogene, is an important component of the dimeric, sequence specific, transcriptional activator, AP-1. Many proteins cooperate with each other in the activation of transcription from specific promoters. Through this cooperation, a gene can be transcribed and a protein product generated. Members of the Fos proto-oncogene family, along with members of the Jun gene family, form stable complexes which bind to DNA at an AP-1 site. The AP-1 site is located in the promoter region of a large number of genes. Binding of the Fos/Jun complex activates transcription of a gene associated with an AP-1 site. In cells that have lost their growth regulatory mechanisms, it is believed that this Fos/Jun complex may "sit" on the AP-1 site, causing overexpression of a particular gene. Since many proliferative disorders result from the overexpression of an otherwise normal gene, such as a proto-oncogene, it would be desirable to identify compositions which interfere with the excessive activation of these genes.
Ras proteins exert their mitogenic and oncogenic effects by activating protein kinase cascades leading to phosphorylation of nuclear transcription factors. AP-1, as described above, is a heterodimeric complex of Jun and Fos proteins, which activates mitogen-inducible genes and is also a major nuclear target of Ras. Ras can stimulate AP-1 activity through c-fos induction (Angel and Karin, Biophys. Acta., 1072:129-157, 1991; Herrlich and Ponta, Trends Genet., 5:112-116, 1989), a process likely to be mediated by the ERK1 and ERK2 mitogen activated protein (MAP) kinases (G. Thomas, Cell, 68:3-6, 1992), through phosphorylation of Elk-1/TCF (Sharrocks, and Shaw, Nature, 358:414-417, 1992; Wynne, et al., Cell, 73:381-393, 1993; Zinck, et al., EMBO J., 12:2377-2387, 1993). However, besides inducing fos and jun gene transcription, mitogens and Ras proteins enhance AP-1 activity through phosphorylation of c-Jun (Binetruy, et al., Nature, 351:122-127, 1991; Smeal, et al, Nature, 354:494-496, 1991; Pulverer, et al., Nature, 353:683-689, 1991). Through an autoregulatory loop, phosphorylation of the c-Jun activation domain leads to c-jun induction (Angel and Karin, supra). Recently, Ras- and UV-responsive protein kinases that phosphorylate c-Jun on serines (Ser) 63 and 73 and stimulate its transcriptional activity were identified (Hibi, et al., Genes & Dev., 7:2135-2148, 1993).
These proline-directed kinases, termed JNK, for c-Jun-N-terminal kinase, are novel MAP kinases (Derijard, et al., Cell, 75:1025-1037, 1994). It is not clear, however, whether c-Jun is the only recipient and whether JNK is the only transducer of the Ras signal to AP-1 proteins. A short sequence surrounding the major JNK phosphorylation site of c-Jun (Ser73) is conserved in c-Fos and is part of its activation domain (Sutherland, et al, Genes & Dev., 6:1810-1819, 1992). The present invention demonstrates that Ras does indeed augment c-Fos transcriptional activity through phosphorylation at Thr232, the homolog of Ser73 of c-Jun. However, this is mediated by a novel Ras- and mitogen-responsive proline-directed protein kinase that is different from JNKs and ERKs.
For many years, various drugs have been tested for their ability to alter the expression of genes or the translation of their messages into protein products. One problem with existing drug therapy is that it tends to act indiscriminately and affects healthy cells as well as neoplastic cells. This is a major problem with many forms of chemotherapy where there are severe side effects primarily due to the action of toxic drugs on healthy cells.
In view of the foregoing, there is a need to identify specific targets in the abnormal cell which are associated with the overexpression of genes whose expression products are implicated in cell proliferative disorders, in order to decrease potential negative effects on healthy cells. The present invention provides such a target.